Effect of pH and buffer on butyric acid production and microbial community characteristics in bioconversion of rice straw with undefined mixed culture

This study was conducted to identify the optimum pH range and the appropriate buffer for butyric acid production from rice straw by fermentation using an undefined mixed culture. A series of experiments conducted at pH levels of 5.0 ~ 7.0 showed that neutral pH improved rice straw conversion and consequently carboxylic acid production. The highest butyric acid production (up to 6.7 g/L) was achieved at pH of 6.0 ~ 6.5, while it was only 1.7 g/L without pH control or at pH 5.0. Another series of experiments conducted at pH 6.0 ~ 6.5 buffered with CaCO₃, NaHCO₃, NH₄HCO₃ and their combinations indicated that different buffers had different effects onthe product spectrum, and that CaCO₃ combined with NaHCO₃ was an effective buffer for butyric acid production. The highest total volatile fatty acids (about 12.6 g/L) production and one of the two highest butyric acid concentrations (about 7.6 g/L) were obtained by buffering with CaCO₃ combined with NaHCO₃. PCR-DGGE analysis revealed that different pH and buffers also influenced the microbial population distribution. Bacteria were suppressed at low pH, while the bacterial community structures at higher pH varied slightly. Overall, this study presents an alternative method for butyric acid production from lignocellulosic biomass without supplementary cellulolytic enzyme.     

A general inhibition kinetics model for ethanol production using a novel carbon source: sodium gluconate

In this study, sodium gluconate was applied as a novel carbon source for the fuel ethanol production using an engineered Escherichia coli strain KO11 in batch fermentations. Ethanol and acetic acid were produced as two major products as well as small amount of lactic acid during the fermentation. Compared to the conventional carbon source glucose, the bioconversion of sodium gluconate possessed two distinct advantages: faster utilization rate of sodium gluconate (1.66 g/L per h) compared to glucose (0.996 g/L per h) and no requirement for pH control during fermentation. A general inhibition model including both substrate and products inhibitory effects was proposed, which adequately simulated batch fermentation kinetics at various concentrations of sodium gluconate. All of the products showed inhibitory effects on cell growth. The order of the inhibitory strength of all products and substrate was for the first time clarified in this study. Acetic acid was the most inhibitory product mitigating the cell growth, followed by ethanol and lactic acid. Sodium gluconate stimulated cell growth when its concentration was below 16 g/L, while it inhibited the cell growth when the concentration was above this concentration. It completely inhibited the cell growth when the concentration was 325 g/L. The high value of both the coefficient of determination (R ²) and the adjusted R ² verified the good fit of the model. This paper provides key insights into further engineering these strains to improve ethanol production.     

CaCO<Subscript>3</Subscript> supplementation alleviates the inhibition of formic acid on acetone/butanol/ethanol fermentation by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis>

Objective

To investigate the inhibiting effect of formic acid on acetone/butanol/ethanol (ABE) fermentation and explain the mechanism of the alleviation in the inhibiting effect under CaCO₃ supplementation condition.

Results

From the medium containing 50 g sugars l^?1 and 0.5 g formic acid l^?1, only 0.75 g ABE l^?1 was produced when pH was adjusted by KOH and fermentation ended prematurely before the transformation from acidogenesis to solventogenesis. In contrast, 11.4 g ABE l^?1 was produced when pH was adjusted by 4 g CaCO₃ l^?1. The beneficial effect can be ascribed to the buffering capacity of CaCO₃. Comparative analysis results showed that the undissociated formic acid concentration and acid production coupled with ATP and NADH was affected by the pH buffering capacity of CaCO₃. Four millimole undissociated formic acid was the threshold at which the transformation to solventogenesis occurred.

Conclusion

The inhibiting effect of formic acid on ABE fermentation can be alleviated by CaCO₃ supplementation due to its buffering capacity.

     

Efficient production of l-lactic acid by newly isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance

A thermophilic Bacillus coagulans WCP10-4 with tolerance to high concentration of glucose was isolated from soil and used to produce optically pure l-lactic acid from glucose and starch. In batch fermentation at pH?6.0, 240 g/L of glucose was completely consumed giving 210 g/L of l-lactic acid with a yield of 95 % and a productivity of 3.5 g/L/h. In simultaneous saccharification and fermentation at 50 °C without sterilizing the medium, 200 g/L of corn starch was completely consumed producing 202.0 g/L of l-lactic acid. To the best of our knowledge, this strain shows the highest osmotic tolerance to glucose among the strains ever reported for lactic acid production. This is the first report of simultaneous saccharification and fermentation of starch for lactic acid production under a non-sterilized condition.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

Optimum conditions for the biological production of lactic acid by a newly isolated lactic acid bacterium,Lactobacillus sp. RKY2

Lactic acid is a green chemical that can be used as a raw material for biodegradable polymer. To produce lactic acid through microbial fermentation, we previously screened a novel lactic acid bacterium. In this work, we optimized lactic acid fermentation using a newly isolated and homofermentative lactic acid bacterium. The optimum medium components were found to be glucose, yeast extract, (NH₄)₂HPO₄, and MnSO₄. The optimum pH and temperature for a batch culture ofLactobacillus sp. RKY2 was found to be 6.0 and 36°C, respectively. Under the optimized culture conditions, the maximum lactic acid concentration (153.9 g/L) was obtained from 200 g/L of glucose and 15 g/L of yeast extract, and maximum lactic acid productivity (6.21 gL⁻¹h⁻¹) was obtained from 100 g/L of glucose and 20 g/L of yeast extract. In all cases, the lactic acid yields were found to be above 0.91 g/g. This article provides the optimized conditions for a batch culture ofLactobacillus sp. RKY2, which resulted in highest productivity of lactic acid.     

Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains

Production of lactic acid from paper sludge was studied using thermophilic Bacillus coagulan strains 36D1 and P4-102B. More than 80% of lactic acid yield and more than 87% of cellulose conversion were achieved using both strains without any pH control due to the buffering effect of CaCO₃ in paper sludge. The addition of CaCO₃ as the buffering reagent in rich medium increased lactic acid yield but had little effect on cellulose conversion; when lean medium was utilized, the addition of CaCO₃ had little effect on either cellulose conversion or lactic acid yield. Lowering the fermentation temperature lowered lactic acid yield but increased cellulose conversion. Semi-continuous simultaneous saccharification and co-fermentation (SSCF) using medium containing 100 g/L cellulose equivalent paper sludge without pH control was carried out in serum bottles for up to 1000 h. When rich medium was utilized, the average lactic acid concentrations in steady state for strains 36D1 and P4-102B were 92 g/L and 91.7 g/L, respectively, and lactic acid yields were 77% and 78%. The average lactic acid concentrations produced using semi-continuous SSCF with lean medium were 77.5 g/L and 77.0 g/L for strains 36D1 and P4-102B, respectively, and lactic acid yields were 72% and 75%. The productivities at steady state were 0.96 g/L/h and 0.82 g/L/h for both strains in rich medium and lean medium, respectively. Our data support that B. coagulan strains 36D1 and P4-102B are promising for converting paper sludge to lactic acid via SSCF.     

Integrated production of lactic acid and biomass on distillery stillage

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L^?1 h^?1 were achieved in batch fermentation with initial sugar concentration of 55 g L^?1. A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10⁹ CFU ml^?1 was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.     

l-Lactic acid production benefits from reduction of environmental osmotic stress through neutralizing agent combination

This paper hinged on the combination effect of two different neutralizing agents Ca(OH)₂ and NH₄OH on the production of l-lactic acid by Lactobacillus paracasei. Present study quantitatively indicated that environmental osmotic pressure (844–1,772 mOsm/kg) exerted minor influence on l-lactic acid production, but a critical level fell on approximately 3,000 mOsm/kg which restricted l-lactic acid production significantly. Once osmotic pressure exceeded 3,600 mOsm/kg, l-lactic acid production ran aground. A new and efficient neutralizing agent-adding strategy was established in this study to procure 2.21-fold enhancement (5.94 g/l/h) relative to previous productivity of l-lactic acid with NH₄OH as neutralizing agent via batch cultivation. It was, therefore, speculated that inhibition effect in the late phase of the fermentation might be in large part attributed to the dramatic increase of environmental osmotic stress, other than cumulative effect of lactate concentration itself.     

Enhancement and modeling of microparticle-added <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactic acid production

Lactic acid has a wide industrial application area and can be produced by fungal strains. However, excessive bulk growth form of fungi during the fermentations is a major problem, which limits the fermentation performance. Microparticles are excellent tools to prevent bulk fungal growth and provide homogenized fermentation broth to increase uniformity and the prediction performance of the models. Therefore, in this study, addition of aluminum oxide and talcum microparticles into fermentations was evaluated to enhance the production of lactic acid by Rhizopus oryzae. The results showed that the bulk fungal growth was prevented and the lactic acid concentration increased from 6.02 to 13.88 and 24.01 g/L, when 15 g/L of aluminum oxide or 10 g/L of talcum was used, respectively, in the shake-flask fermentations. Additionally, substrate concentration, pH, and agitation were optimized in the bioreactors using response surface methodology, and optimum values were determined as 126 g/L of glucose, 6.22 pH, and 387 rpm, respectively. Under these conditions, lactic acid production further increased to 75.1 ± 1.5 g/L with 10 g/L of talcum addition. Also, lactic acid production and glucose consumption in the batch fermentation were successfully modeled with modified Gompertz model and modified logistic model. RMSE and MAE values for lactic acid production were calculated as 2.279 and 1.498 for the modified Gompertz model; 3.6 and 4.056 for the modified logistic model. Additionally, modified logistic model predicted glucose consumption with ?2.088 MAE and 2.868 RMSE, whereas these values were calculated as 2.035 and 3.946 for the modified Gompertz model.     

Optimization of lactic acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130

Production of lactic acid from beet molasses by Lactobacillus delbrueckii NCIMB 8130 in static and shake flask fermentation was investigated. Shake flasks proved to be a better fermentation system for this purpose. Substitution of yeast extract with other low cost protein sources did not improve lactic acid production. The maximum lactic acid concentration was achieved without treatment of molasses. A Central Composite Design was employed to determine the maximum lactic acid concentration at optimum values for the process variables (sucrose, yeast extract, CaCO₃). A satisfactory fit of the model was realized. Lactic acid production was significantly affected both by sucrose–yeast extract and sucrose–CaCO₃ interactions, as well as by the negative quadratic effects of these variables. Sucrose and yeast extract had a linear effect on lactic acid production while the CaCO₃ had no significant linear effect. The maximum lactic acid concentration (88.0 g/l) was obtained at concentrations for sucrose, yeast extract and CaCO₃ of 89.93, 45.71 and 59.95 g/l, respectively.     

A novel isolate of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h^?1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.     

Lactic acid and animal feeds production from Sophora flavescens residues by Rhizopus oryzae fermentation

In this study, the feasibility of producing lactic acid and animal feeds from Sophora flavescens residues (SFR) by Rhizopus oryzae was explored. Results showed that the simultaneous saccharification and fermentation (SSF) is the optimal fermentation mode, which was simple and high-efficiency. When the inoculation volume of R. oryzae was 10 % and the pH value was adjusted by adding CaCO₃ in stages during SSF, the maximum concentration of lactic acid was 46.78 g/L, and the maximum lactic acid productivity reached 0.97 g/L/h. Results also showed that the protein content of the solid residues after fermentation of R. oryzae reached 12.15 %. This content was 46 times higher than that by the original SFRs and nearly 4.3 times the protein content of the solid residues after fermentation by Enterococcus faecium. In addition, the solid residues after fermentation rich in Fe and Zn could be used as animal feeds or feed additives. Thus, it is expected that this study may provide a novel approach for Chinese medicine residues treatment towards full resource recovery.     

Utilization of by-products derived from bioethanol production process for cost-effective production of lactic acid

The by-products of bioethanol production such as thin stillage (TS) and condensed distillers solubles (CDS) were used as a potential nitrogen source for economical production of lactic acid. The effect of those by-products and their concentrations on lactic acid fermentation were investigated using Lactobacillus paracasei CHB2121. Approximately, 6.7 g/L of yeast extract at a carbon source to nitrogen source ratio of 15 was required to produce 90 g/L of lactic acid in the medium containing 100 g/L of glucose. Batch fermentation of TS medium resulted in 90 g/L of lactic acid after 48 h, and the medium containing 10 % CDS resulted in 95 g/L of lactic acid after 44 h. Therefore, TS and CDS could be considered as potential alternative fermentation medium for the economical production of lactic acid. Furthermore, lactic acid fermentation was performed using only cassava and CDS for commercial production of lactic acid. The volumetric productivity of lactic acid [2.94 g/(L·h)] was 37 % higher than the productivity obtained from the medium with glucose and CDS.     

Fermentative production of shikimic acid: a paradigm shift of production concept from plant route to microbial route

Different physiological and nutritional parameters affect the fermentative production of shikimic acid. In our study, Citrobacter freundii initially produced 0.62 g/L of shikimic acid in 72 h. However, when process optimization was employed, 5.11 g/L of shikimic acid was produced in the production medium consisting of glucose (5.0 %), asparagine (4.5 %), CaCO₃ (2.0 %), at pH 6.0, when inoculated with 6 % inoculum and incubated at 30 ± 1 °C, 200 rpm for 60 h. Preliminary fed-batch studies have resulted in the production of 9.11 g/L of shikimic acid on feeding the production medium by 20 g/L of glucose at 24 h of the fermentation run. Production of similar amount of shikimic acid was observed when the optimized conditions were employed in a 10-L bioreactor as obtained in shake flask conditions. A total of 9.11 g/L of shikimic acid was produced in 60 h. This is approximately 14.69-fold increase in shikimic acid production when compared to the initial un-optimized production conditions. This has also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Co-production of lactic acid and chitin using a pelletized filamentous fungus Rhizopus oryzae cultured on cull potatoes and glucose

Aims: This paper developed a novel process for lactic acid and chitin co-production of the pelletized Rhzious oryzae NRRL 395 fermentation using underutilized cull potatoes and glucose as nutrient source. Methods and Results: Whole potato hydrolysate medium was first used to produce the highest pelletized biomass yield accompanying the highest chitin content in biomass. An enhanced lactic acid production then followed up using batch, repeated batch and fed batch culture with glucose as carbon source and mixture of ammonia and sodium hydroxide as neutralizer. The lactic acid productivity peaked at 2·8 and 3 g l⁻¹ h⁻¹ in repeated batch culture and batch culture, respectively. The fed batch culture had the highest lactate concentration of 140 g l⁻¹. Conclusions: Separation of the biomass cultivation and the lactic acid production is able to not only improve lactic acid production, but also enhance the chitin content. Cull potato hydrolysate used as a nutrient source for biomass cultivation can significantly increase both biomass yield and chitin content. Significance and Impact of the Study: The three-step process using pelletized R. oryzae fermentation innovatively integrates utilization of agricultural residues into the process of co-producing lactic acid and chitin, so as to improve the efficiency, revenues and cost of fungal lactic acid production.     

Improvement of mannitol production by Lactobacillus brevis mutant 3-A5 based on dual-stage pH control and fed-batch fermentations

Lactobacillus brevis 3-A5 was isolated and expected to produce mannitol efficiently by regulating pH in batch and fed-batch fermentations. In 48 h batch fermentations with free and constant pH, the optimal pH for cell growth and mannitol production in the first 24 h of incubation was 5.5, whereas that for mannitol production in the second 24 h of incubation was 4.5. To achieve high cell density and mannitol yield simultaneously, a dual-stage pH control strategy was proposed based on the kinetic analysis of mannitol production. The pH value was controlled at 5.5 for the first 12 h of fermentation and subsequently shifted to 4.5 until the fermentation was completed. Under dual-stage pH control fermentation, a 103 g/L yield of mannitol with a volumetric production rate of 3.7 g/L/h was achieved after 28 h. The dual-stage pH control fed-batch fermentation strategy was further developed to improve mannitol yield, wherein the yield increased by 109 % to 215 g/L after 98 h of fermentation. This value is the highest yield of mannitol ever reported using L. brevis.